The mechanism of insulin action on gene expression is a key question in biology, with important ramifications for the treatment of diabetes and metabolic disorders. Studies supported by this grant have established a role for the O sub-family of Forkhead transcription factors in insulin regulation of gene expression. During the past funding cycle, we have demonstrated that FoxO1 is the principal insulin-dependent transcription factor in the regulation of hepatic gluconeogenesis and pancreatic beta cell mass. We have shown that: i, phosphorylation is the main mechanism by which insulin inhibits FoxO1 by promoting its nuclear exclusion; ii, FoxO1 expression can single-handedly confer insulin regulation on the expression of Glucose-6-phosphatase, the rate-limiting enzyme in glycogenolysis; iii, FoxO1 interacts with Pgc1alpha to stimulate gluconeogenesis; [unreadable] iv, FoxO1 links insulin signaling to Pdx1 regulation of beta cell mass. We now present preliminary data extending the gamut of FoxO functions to regulation of cell differentiation and protection against oxidative stress, while also expanding the repertoire of FoxO target genes. We demonstrate that these functions are based on two novel modes of FoxO action: acetylation-dependent 'targeting to nuclear Pml bodies, and protein/protein interactions with the Notch effector Csl. The latter observation indicates that FoxO1 functions as a coactivator, and not only as a transcription factor. It also bridges together two important signaling pathways, the PI 3-kinase and the Hes1 pathways. In the next five years, we will endeavor to integrate this new information in the mechanism of FoxO1 action and its role in metabolic disorders. We propose to study: in Aim 1, how phosphorylation- and acetylation-mediated mechanisms are integrated in vivo to determine the kinetics of FoxO1 sub-cellular localization in physiologic conditions and disease states; in Aim 2, how acetylation-dependent sub-nuclear targeting of FoxO1 regulates metabolism in liver, pancreatic beta cells and adipocytes; and in Aim 3, how the balance between the coactivator and transcription functions of Foxo1 is determined. The studies will be carried out with genetic loss- and gain-of-function experiments in transgenic mice and cultured cells, using methods that have been fully implemented in the PI's laboratory. The ultimate goal of this work is to find a therapeutic approach to modifying FoxO1 function. Indeed, while FoxO1 is an extremely attractive biological target to treat diabetes and metabolic diseases, it is largely intractable as a drug target. Therefore, it is hoped that by identifying mechanisms of action and interacting partners, new ways to modulate its function can be found. [unreadable] [unreadable]